Single Vesicle Millisecond Fusion Kinetics Reveals Number of SNARE Complexes Optimal for Fast SNARE-mediated Membrane Fusion*

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca2+, and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.

[1]  L. Tamm,et al.  Clustering of syntaxin-1A in model membranes is modulated by phosphatidylinositol 4,5-bisphosphate and cholesterol. , 2009, Biochemistry.

[2]  Antoine M. van Oijen,et al.  Single-particle kinetics of influenza virus membrane fusion , 2008, Proceedings of the National Academy of Sciences.

[3]  J. Rizo,et al.  Synaptic vesicle fusion , 2008, Nature Structural &Molecular Biology.

[4]  R. Jahn,et al.  Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids , 2007, Nature Structural &Molecular Biology.

[5]  Thorsten Lang,et al.  Anatomy and Dynamics of a Supramolecular Membrane Protein Cluster , 2007, Science.

[6]  Helmut Grubmüller,et al.  Molecular Anatomy of a Trafficking Organelle , 2006, Cell.

[7]  L. Tamm,et al.  Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking. , 2006, Biophysical journal.

[8]  Reinhard Jahn,et al.  SNAREs — engines for membrane fusion , 2006, Nature Reviews Molecular Cell Biology.

[9]  Alexander Stein,et al.  N- to C-Terminal SNARE Complex Assembly Promotes Rapid Membrane Fusion , 2006, Science.

[10]  Edwin R Chapman,et al.  Ca2+–synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion , 2006, Nature Structural &Molecular Biology.

[11]  Edwin R Chapman,et al.  SNARE-driven, 25-millisecond vesicle fusion in vitro. , 2005, Biophysical journal.

[12]  S. Pantano,et al.  SNARE complexes and neuroexocytosis: how many, how close? , 2005, Trends in biochemical sciences.

[13]  L. Tamm,et al.  Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[14]  A. Brunger,et al.  Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs). , 2004, Biophysical journal.

[15]  J. Rothman,et al.  Imaging single membrane fusion events mediated by SNARE proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Jackson,et al.  Transmembrane Segments of Syntaxin Line the Fusion Pore of Ca2+-Triggered Exocytosis , 2004, Science.

[17]  C. Seidel,et al.  Determinants of liposome fusion mediated by synaptic SNARE proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Dirk Fasshauer,et al.  A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly* , 2004, Journal of Biological Chemistry.

[19]  R. Schneggenburger,et al.  Presynaptic Capacitance Measurements and Ca2+ Uncaging Reveal Submillisecond Exocytosis Kinetics and Characterize the Ca2+ Sensitivity of Vesicle Pool Depletion at a Fast CNS Synapse , 2003, The Journal of Neuroscience.

[20]  L. Donald Partridge,et al.  Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis , 2002, Nature Neuroscience.

[21]  M. L. Wagner,et al.  Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers. , 2001, Biophysical journal.

[22]  R. Scheller,et al.  Three SNARE complexes cooperate to mediate membrane fusion , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. L. Wagner,et al.  Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker. , 2000, Biophysical journal.

[24]  Ralf Schneggenburger,et al.  Intracellular calcium dependence of transmitter release rates at a fast central synapse , 2000, Nature.

[25]  W. Antonin,et al.  Mixed and Non-cognate SNARE Complexes , 1999, The Journal of Biological Chemistry.

[26]  Reinhard Jahn,et al.  Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution , 1998, Nature.

[27]  W. Xiao,et al.  The synaptic SNARE complex is a parallel four-stranded helical bundle , 1998, Nature Structural Biology.

[28]  Benedikt Westermann,et al.  SNAREpins: Minimal Machinery for Membrane Fusion , 1998, Cell.

[29]  B. Sakmann,et al.  Calcium influx and transmitter release in a fast CNS synapse , 1996, Nature.

[30]  S. Durell,et al.  Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events , 1996, The Journal of cell biology.

[31]  P. Hanson,et al.  Botulinum and tetanus neurotoxins: emerging tools for the study of membrane fusion. , 1995, Cold Spring Harbor symposia on quantitative biology.

[32]  Thomas C. Südhof,et al.  Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25 , 1993, Nature.

[33]  Paul Tempst,et al.  SNAP receptors implicated in vesicle targeting and fusion , 1993, Nature.

[34]  L. Tamm,et al.  Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers. , 1992, Biochimica et biophysica acta.

[35]  H. Mcconnell,et al.  Supported phospholipid bilayers. , 1985, Biophysical journal.

[36]  H. Akaike.  A new look at the statistical model identification , 1974 .

[37]  A. Hodgkin,et al.  A quantitative description of membrane current and its application to conduction and excitation in nerve , 1952, The Journal of physiology.